1. Field of the Invention
The present invention in the field of biology and medicine relates to a cell culture model system that mimics human preneoplastic breast disease and uses thereof in screening agents that inhibit development of this disease and its progression to breast cancer.
2. Description of the Background Art
Reciprocal cellular interactions between epithelial and stromal cells have been demonstrated as a key determinant in the morphogenesis, proliferation and cyto-differentiation of both endocrine and non-endocrine target organs (I. Hom, Y. K. et al., Endocrinology, 139: 913-921, 1998; Donjacour, A. A. et al., Cancer Treatment Res., 53: 335-364, 1991; Cunha, G. R. et al., Cell Differ., 17:137-148, 1985). Carcinomas of the breast are composed of not only tumor epithelial cells but also of infiltrating endothelial cells (xe2x80x9cEC""sxe2x80x9d), fibroblasts, macrophages and lymphocytes (Gregoire, M. et al., Cancer Metast. Rev., 14: 339-350, 1995). The stroma provides vascular supply and specific soluble and extracellular matrix (ECM) molecules that are required for tumor growth and progression (Hanahan, D. et al., Cell, 86: 353-364, 1996). Several lines of evidence indicate that stromal cells play a central role via ECM remodeling in tumor invasion and dissemination (Camps, J. L. et al., Proc. Natl. Acad., Sci. USA, 87: 75-79, 1990; Picard, O., et al. Cancer Res., 46: 3290-3294, 1986; Grey, A. M. et al., Proc. Natl. Acad. Sci. USA, 86: 2438-2442, 1989). However, a recent report has shown that stromal alteration(s) precede the malignant conversion of tumor cells (Moinfar, F. et al., Cancer Res., 60: 2562-2566, 2000).
Although there is experimental evidence supporting the involvement of angiogenesis in pathogenesis of breast cancer, the influence of functional interactions between human breast epithelial cells (also referred to as mammary epithelial cells) and endothelial cells have not been defined. Analysis of such cell interactions requires a culture/assay system that permits growth and differentiation of both epithelial cells and endothelial cells.
The present invention provides such a system and describes its utility as a model for studying the progress of preneoplastic lesions to cancer and for testing the activity of agents that can inhibit this process.
Growth and formation of capillary blood vessels or neovascularization is an essential component of solid tumor growth (Folkman, J. et al., Int. Rev. Exp. Pathol. 16: 207, 1976; Gimbrone, M. A. Jr et al., J. Natl. Cancer Inst., 52: 413, 1974). Any increase in the size of the tumor cell population must be preceded by an increase in new capillaries that converge upon the tumor; such angiogenesis has been directly correlated with tumor growth and metastasis (Folkman, J, J. Nat""l Canc Inst 82:4-6, 1990). Tumor cell products and products of various non-neoplastic mediator systems have been implicated in this vasoproliferative response (Gimbrone et al., supra; Auerbach, R., In: E. Pick (ed), Lymphokines. Vol 4, pp. 69-84, New York: Academic Press, 1981). Several growth factors, cytokines, molecules of the extracellular matrix (ECM) or physical conditions induce or regulate endothelial cell growth and/or migration in vitro (Klagsbrun, M. et al., Ann. Rev. Physiol. 53:217-239, 1991). These include several well characterized polypeptide growth factors, proteolytic enzymes, interferon, cyclic nucleotides, prostaglandins, heparin, lowered oxygen tension, histamine and other vasoactive amines, and several low molecular weight endothelial mitogens and chemotactic factors (Klagsbrun et al., supra).
Vascular endothelial growth factor (Ferrara, N. et al., Biochem. Biophys. Res. Commun., 161:851-858, 1989) or vascular permeability factor (Connolly, D. T. et al., J. Biol. Chem., 264:20017-20024, 1989) (abbreviated VEGF/PF or VEGF) is an endothelial cell-specific mitogen that mediates physiological and pathological neovascularization (Leung, D. W. et al., Science 246:1306-1309, 1989). VEGF acts as a survival factor, preventing the apoptotic death of microvascular endothelial cells (Alon, T. et al., Nat. Med., 1:1024-1028, 1995, 1995; Watanabe, Y. et al., Exp. Cell. Res., 233:340-349, 1997). The human VEGF gene encodes a dimeric glycoprotein comprising four possible monomers as a result of differential splicing of eight exons that make up the gene product. The four VEGF subtypes are 121-, 165-, 189-, and 206-amino acids in length (Neufeld, G. et al., FASEB J., 13:9-22, 1999). The smaller forms are secreted whereas VEGF189 and VEGF206 are bound to heparan proteoglycans and thus retained close to the membrane of producing cells. Three receptors for VEGF have been described:
VEGFR-1 (=Flt-1) binds VEGF;
VEGFR-2 (=Flk-1/KDR) binds VEGF; and
VEGFR-3 (=Flt-4) appears to be specific for VEGF-C (Neufeld et al., supra).
Expression of Flk-1/KDR is confined to endothelial cells, accounting for the selective nature of VEGF-induced mitogenesis (Neufeld, supra). VEGF is expressed at high levels in a wide range of tumors and tumor cell lines (Berse, B. et al., Mol. Biol. Cell, 3:211-220, 1992) and is believed to be a key mediator of (1) tumor angiogenesis (Connolly, D. T. et al., J. Clin. Invest., 84:1470-1477, 1989; Kim, K. J. et al., Nature (London), 362:841-844, 1993; Plate, K. H. et al., Nature (London), 359:845-848, 1992) and (2) the high blood vessel permeability characteristic of tumors (Senge, D. R. et al., Science 219:983-985, 1983; Yeo, K. T. et al., Cancer Res., 53:2912-2918, 1983). Expression of VEGF in the uterus was rapidly and strongly stimulated by estrogen (Cullinan-Bove, K. et al., Endocrinology, 133:829-837, 1993), suggesting that VEGF mediates the normal, estrogen-induced increase in vascular permeability and blood vessel growth in the uterus. Similarly, expression of VEGF is rapidly induced by 17 xcex2-estradiol (E2) in dimethylbenzanthracene (DMBA)-induced estrogen-dependent mammary tumors (Nakamura, J. et al., Endocrinology, 137:5589-5596, 1996).
Using the MCF10AT1 xenograft model for human proliferative breast disease, the present inventors and their colleagues previously demonstrated that E2 exerts a growth promoting effect on benign or premalignant ductal epithelium by enhancing the speed of transformation from simple/mild hyperplasia (grades 0/1) to atypical hyperplasia (grade 3) and ductal carcinoma in situ (grade 4) (Shekhar, P. V. M. et al., Amer. J. Pathol., 152:1129-1132, 1998). Table 1, below, summarizes the criteria for grading proliferative breast lesions (from Dawson, P. J. et al., Am. J. Pathol., 148:313-319, 1996).
Much of this growth promoting effect appeared to arise from effects of E2 on angiogenesis since lesions from unsupplemented animals were either simple or hyperplastic without atypia and lack angiogenesis. The dramatic increase in growth and advanced histological grades of progression concomitant with its remarkable effect on angiogenesis suggested to the present inventors that one of the mechanisms by which E2 acts as a breast cancer promoter could be through its effect on expression of angiogenesis-regulating factors.
The extracellular matrix (ECM) acts locally to modulate the responsiveness of endothelial cells and mammary epithelial cells to external factors. Besides providing a scaffolding during capillary morphogenesis, the ECM, by virtue of its ability to mediate both biochemical and biomechanical signaling events, exerts complex local controls on the functions of endothelial cells (Polverini, P. J., Eur. J. Cancer, 32A:2430-2437, 1996). For example, the ECM controls growth, differentiation and apoptosis of normal murine and human breast epithelial cells (Barcellos-Hoff, M. H. et al., Development, 105:223-235, 1989; Boudreau, N. et al., Science 267:891-893, 1995).
Collagenolytic degradation of endothelial and parenchymal basement membranes is an essential step in the process of tumor invasion and angiogenesis (Liotta, L. A. et al., Cell, 64:327-336, 1991). Proteolysis and interruption of the basement membrane and ECM require the activation of specialized matrix metalloproteinases (MMPs), the type IV collagenases or gelatinases that degrade basement membrane collagens type IV and V (Liotta, L. A. et al., Nature 284:67-68, 1980). Two MMP species have been cloned and sequenced:
(1) the 72 kDa enzyme known as MMP-2 and gelatinase A) and
(2) the 92 kDa enzyme known as MMP-9 and gelatinase B
(Liotta et al., supra, Liotta, L. A. et al., Biochemistry, 20:100-104, 1981; Wilhelm, S. M. et al., J. Biol. Chem., 264:17213-21, 1989). MMP-2 and MMP-9 are secreted as latent proenzymes; activation requires removal of an 80 residue and 87 residue N-terminal domain, respectively (Stetler-Stevenson, W. G. et al., J. Biol. Chem., 264:1353-6, 1989; Collier, I. E. et al., J. Biol. Chem., 263:6579-87, 1988).
Stampfer et al., U.S. Pat. No. 4,423,145 describes growth medium and conditions for culturing human mammary epithelial cells. The document discloses that clonal growth of these cells is improved by using a skin fibroblast feeder layer. Although this is an example of a two cell heterotypic culture, it differs from the present invention in numerous significant ways, in that no mention is made of co-culture with endothelial cells. Moreover, in contrast to the present invention fibroblasts are used merely as a feeder layer to allow better proliferation of epithelial cells that are plated at low density for cloning. Under these conditions, although the epithelial cells proliferate better, they do not undergo any morphological conversions such as those described in the present invention. Furthermore, skin fibroblasts, which are not part of the present invention, are the only fibroblast type disclosed.
Naughton et al., U.S. Pat. No. 5,032,508 discloses a 3D matrix and its use as the framework for a 3D, multi-layer cell culture system which is said to be an improvement over previously known tissue culture systems wherein cells grew in a monolayer. In this approach, a solid phase 3D support material that allows cells to attach and to grow in more than one layer is inoculated with stromal cells, comprising fibroblasts with or without additional cells. This stromal matrix (which can be generic or tissue-specific) is then inoculated with parenchymal or tissue cells which grow the 3D stromal support in multiple layers, forming a cellular matrix. This matrix system was said to be a closer model of physiologic in vivo conditions than were previously described monolayer tissue culture systems. The 3D cell culture system was disclosed as being applicable to proliferation of different types of cells and the formation of a number of different tissues. The document specifically listed and exemplified bone marrow, skin, liver, pancreas, kidney, adrenal and neurologic tissue. This system was said to be useful for in vitro cytotoxicity testing and screening compounds including growth/regulatory factors, pharmaceutical agents. This document does not disclose breast epithelial cells, and only mentions mucosal epithelium (model systems to study herpesvirus or papilloma virus infection) or dermal epithelium in modeling skin. The only mention of xe2x80x9cbreastxe2x80x9d is in the background where the document refers to 3D collagen gels for culturing breast epithelium (Yang et al., 1981, Cancer Res. 41:1021-1027). Breast tissue, breast epithelial cells, breast fibroblasts or breast disease are never mentioned in the description of the invention. Endothelial cells are discussed only as one type of stromal cell, e.g., in vascular endothelium (for modeling blood brain barrier in vitro) bile duct endothelium (liver culture) or in bone marrow cultures.
Jones et al., U.S. Pat. No. 5,935,853, discloses the discovery that the MCF-10A cell line, an immortalized human mammary epithelial cell line, produces an ECM which is capable of stimulating hemidesmosome formation in unrelated epithelial cells contacted with the matrix. The MCF-10A cell line produces both a deposited (insoluble) and a similar secreted (soluble) matrix. The document discloses a method of growing epithelial cells by contacting the cells with the ECM deposited or secreted MCF-10A cells.
Chang et al., U.S. Pat. No. 5,814,511 describes a substantially purified human breast epithelial cell (Type I HBEC) and a method of obtaining these epithelial cells comprising the steps of: a) development of a mixture of human breast epithelial cells from reduction mammoplasty tissues using the MSU-1 medium; b) eliminating stromal fibroblasts by a trypsin (0.002%) and ethylenediamine tetraacetic acid (0.02%) solution; c) separating Type I HBEC from Type II HBEC which attach on culture dishes earlier by collecting Type I HBEC that remain in suspension after trypsinization and prolonged incubation; d) the continuing culture of these cells in MSU-1 medium supplemented with fetal bovine serum, which inhibits the growth of Type II HBEC while promoting the growth of Type I HBEC, gives rise to Type I HBEC. Described also is a new defined medium (the MSU-1 medium) which supports the growth of both Type I and Type II human breast epithelial cells.
Goodwin et al., U.S. Pat. Nos. 5,308,764 and 5,496,722, disclose 3D cell cultures comprising aggregates of normal mammalian cells (for each of the three major tissue groups). The culture aggregates were produced under microgravity culture conditions (microgravity or simulated microgravity created in unit gravity by controlling the horizontal rotation of a culture vessel containing normal mammalian cells). These cell aggregates exhibited 3D tissue growth and functional interrelationship by cell to cell contact. Functional cells with normal morphology were produced for organ, structural and blood producing tissues. The process for producing the normal mammalian tissue was said to be is particularly unique in that it could produce normal tissue of 2 mm and larger. The starting cell inoculum was predominantly normal differentiated epithelial cells and predominantly normal differentiated mesenchymal cells (disassociated prior to introduction). The epithelial and mesenchymal cells were introduced in a vessel with a culture matrix preferably of generally spherical microcarriers. Tissue engineering was achieved by selected introduction of the mesenchymal cells and culture matrix for a preselected culture period prior to transfer of epithelial cells to the culture vessel.
Goodwin et al., U.S. Pat. No. 5,153,132 discloses a method for culturing at least two distinct originating types of mammalian cells such as stromal cells and epithelial cells. The method, which requires a constantly and controllably rotating culture chamber and the presence of microcarriers (cell attachment substrates), resulted in the in vitro generation of multi-cellular, 3D, differentiated, organized living tissues.
Collier et al., U.S. Pat. No. 5,059,586 disclosed enhancement of the growth of milk-producing mammary parenchyma in a mammal by intramammary infusion a substance that was mitogenic for mammary epithelial cells in the mammal.
Archer et al., U.S. Pat. No. 4,439,521, disclosed a method for producing 3_D pancreatic islet-like structures having histology and functionality (insulin-production) corresponding to those of fetal pancreatic islets and islets from adult animals maintained in culture. The method involved culturing, attached to a substrate, isolated natural pancreatic islets, pancreatic duct pieces, cell clusters consisting of mildly digested pieces of pancreas, cell tissues obtained as by-products of the culturing methods, or previously-produced islet-like structures. The formation of the structures occurs over about 5-17 weeks in culture. This document does not disclose the 3D culture of any other types of cells.
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.
Since commitment to the morphogenetic and differentiation programs requires the establishment of intercellular communication between breast stromal and epithelial cells, the present inventors have established a novel three dimensional (xe2x80x9c3Dxe2x80x9d) cell-cell interaction model to study the molecular and cellular basis of epithelial-fibroblast-endothelial cell interactions.
The present model/assay system recapitulates in vitro the in vivo processes that lead to breast cancer development and progression from preneoplastic tissue. This model is the first that allows demonstration in vitro the de novo development and neoplastic conversion of functional alveolar units. Advantages of this model include the fact that it requires only about 7 days for alveolar morphogenesis to occur compared to 8-10 weeks in vivo. This permits the use of this system for relatively high throughput drug screening (see below).
As described below, this system has been used to compare the abilities of specific mesenchymal cell types and human umbilical vein endothelial cells (HUVEC) to induce three dimensional morphogenesis and growth of normal-behaving MCF10A and preneoplastic MCF10AT1-EIII8 human breast epithelial cells. The present invention demonstrates not only a requirement for breast-specific fibroblasts but also shows the dominant manner by which normal breast fibroblasts (obtained during reduction mammoplasty) and tumor-derived breast fibroblasts suppress or induce, respectively, growth and ductal-alveolar morphogenesis of MCF10A and MCF10AT1-EIII8 breast epithelial cells.
In this model, preneoplastic human breast epithelial cells interact with two major stromal components, endothelial cells and fibroblasts, on a reconstituted basement membrane and undergo alveolar morphogenesis, a critical step in breast tissue morphogenesis. The assay system shows that alveolar morphogenesis of human breast epithelial cells occurs when the preneoplastic epithelial cells interact with either endothelial cells or breast fibroblasts. However, neoplastic conversion of alveolar functional units occurs only when endothelial cells are present in epithelial-fibroblast co-cultures. The present inventors have shown for the first time using this model the biological requirements and/or contribution from epithelial cells and stromal components for formation of functional ductal lobular units, and processes that allow neoplastic conversion.
Thus, the present invention is directed to a 3D in vitro culture system that serves as a model for the development and progression of preneoplastic breast disease and is therefore useful for screening therapeutic agent that prevent or inhibit breast cancer development, the model system comprising a co-culture of
(a) preneoplastic breast epithelial cells; (b) endothelial cells; and (c) breast fibroblasts on a reconstituted basement membrane in the presence of medium containing (i) effective concentrations of growth factors and additives that act on the epithelial and endothelial cells, and (ii) effective concentrations of an estrogen such that the cells undergo morphogenesis that results in the formation of s multicellular 3D network of branching ductal alveolar units in culture within about 3-7 days. The above cells are preferably human cells.
In this culture system, the growth factors acting on endothelial cells preferably comprise epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and fibronectin, and the growth factors and additives acting on the epithelial cells preferably comprise cholera toxin, insulin, EGF and hydrocortisone.
In the above culture system, the epithelial cells are preferably transformed by T24 Ha-ras cells or are cells derived therefrom by xenotransplantation in nude mice. Preferred lines of epithelial cells are MCF10AT1 or MCF10AT1-EIII8 (xe2x80x9cEIII8xe2x80x9d) cells, in particular EIII8.
In the above culture system, the endothelial cells are preferably human umbilical vein endothelial cells (HUVEC and the reconstituted basement membrane is preferably Matrigel(copyright).
A preferred medium for the above culture system is DMEM-F12 medium, which more preferably is supplemented with 0.1 xcexcg/ml cholera toxin, 10 xcexcg/ml insulin, 0.5 xcexcg/ml hydrocortisone, 0.02 xcexcg/ml EGF.
In the above culture system 1 wherein the preferred estrogen is estradiol at a concentration between about 1 and 10 nM.
The epithelial cells in the above culture system preferably produce mucin and express cytokeratins and proliferating cellular nuclear antigen.
In the above culture system, as a result of secretion by the cells, the medium preferably contains measurable concentrations or activities of one or more of interleukin-8, matrix metalloproteinase-2 and VEGF.
In another embodiment, the present invention provides a method for assaying a test agent for its activity of preventing or inhibiting the development or progression of preneoplastic breast disease. The method comprises:
(a) adding the test agent to a culture that comprises the model system of claim 1 for an interval sufficient for the agent to act upon the preneoplastic breast epithelial cells, the endothelial cells or the fibroblasts
(b) to a parallel culture, adding a negative control agent for the interval, which negative control agent does not prevent or inhibit the progression,
(c) examining the cultures of (a) and (b), above, for
i. formation of the branching ductal alveolar units by morphology; or
ii. proliferation of cells; or
iii. the presence in the culture medium of one or more secreted products of the cells
wherein prevention or inhibition of the unit formation, the proliferation or generation of the secreted products in cultures of (a) compared to (b), is indicative that the agent has the activity.
The above method may also include the addition of a third parallel culture group which is a positive control agent known to inhibit the formation, proliferation or presence of secreted products.
In one embodiment of the above method, the assaying comprises, in step (c), examining the cultures morphologically for the branching ductal alveolar units. Another embodiment measures the proliferation of cells, preferably by a colorimetric assay. Yet another embodiment measures the presence or amount of the secreted products in the culture medium, for example, by assaying immunoreactivity in an immunoassay or by biological activity in a bioassay. Examples of such secreted products included growth or angiogenic factors, e.g., VEGF.
The secreted factor may be assayed for its stimulation of proliferation or of endothelial tube formation (thought to be related to angiogenesis) in culture.
In one embodiment, the test agent inhibits proteolytic enzymes that are required for invasion and transformation to malignancy. Thus, the secreted product may be a matrix metalloproteinase that is assayed by enzymatic activity on a specific substrate.
The test agent may be one that induces terminal differentiation of breast epithelial cells and thereby inhibits neoplastic conversion.
The present invention also is directed to a method for testing an agent for its activity as an endothelial cell-specific or epithelial cell-specific factor active in promoting ductal-alveolar morphogenesis, angiogenesis and progression of preneoplastic breast epithelial cells to a malignant phenotype, comprising:
(a) adding the agent to a culture that comprises the model system of claim 1 for an interval sufficient for the agent to act upon the preneoplastic breast epithelial cells or the endothelial cells;
(b) to a parallel culture, adding a negative control agent for the interval, which negative control agent does not promote ductal-alveolar morphogenesis, angiogenesis or progression of preneoplastic breast epithelial cells to a malignant phenotype;
(c) examining the cultures of (a) and (b), above, for
i. formation of the branching ductal alveolar units by morphology; or
ii. cellular changes corresponding to angiogenesis; or
iii. progression of preneoplastic breast epithelial cells to a malignant phenotype,
wherein promoting of the unit formation, the cellular changes corresponding to angiogenesis, or the progression in cultures of (a) compared to (b), is indicative that the agent has the activity.
In the above method, it is useful to add a third parallel culture group which is a positive control agent known to promote ductal-alveolar morphogenesis, angiogenesis or progression of preneoplastic breast epithelial cells to a malignant phenotype.